1b.Approach (from AD-416)
Selected tissues from sweet potato and its diploid relatives will be collected
and mRNA isolated and converted to cDNA to be sequenced by 454 technology at ARS
Location.

3.Progress Report

The Genomics and Bioinformatics Research Unit does not have the necessary facilities to grow plant material under different growing conditions nor does it have expertise in sweet potato physiology, whereas the scientists at Alcorn State University (ASU) specialize in sweet potato. Various sweetpotato tissue samples have been collected and RNA isolated at the Biotech Center, ASU for the project. In total 43 samples were used to generate a normalized cDNA library and which were subjected to two full runs on the Roche GS-FLX for DNA sequencing.

The developmental stages were:

1. Young, middle-aged, and old aged leaves from hydroponic plants under high or low nitrogen media (6 samples).
2. Leaves of similar age from hydroponic plants with or without treatment of Apogee under low or high nitrogen media (5 samples).
3. Mature leaves from field-grown plants (1 sample).
4. Cold challenged or frosted leaves, leaf blade or petioles from field or pot-grown plants (5 samples).
5. Leaves from drought challenged plants in pots (2 samples).
6. Young, middle, and old vines from hydroponic plants under high or low nitrogen media (9 samples).
7. Vines of similar age from hydroponic plants with or without treatment of Apogee under low or high nitrogen media (4 samples).
8. Vines from field-grown plants (1 sample).
9. Cold challenged or frosted vines from field or pot-grown plants (2 samples).
10. Vines from drought challenged plants in pots (2 samples).
11. Young, middle-aged, and old Fibrous roots from hydroponic plants under high or low nitrogen media (3 samples).
12. Fibrous roots from hydroponic plants with or without treatment of Apogee under low or high nitrogen media (4 samples).
13. Pencil roots from hydroponic plants with or without treatment of Apogee under low or high nitrogen media (4 samples).
14. Storage roots from filed-grown plants with or without treatment of Apogee (3 samples).
15. Cured storage roots (1 sample).
16. Storage of different developmental stages from hydroponic grown plants (3 samples).

In addition, total RNA samples were prepared in large-scale (>1mg) from various tissues of a diploid and a tetraploid progenitor to the hexaploid sweetpotato, Ipomoea tenuissima (2x) and Ipomoea. littoralis (4x). The RNA samples from each species were combined for making a normalized cDNA library, and for sequencing (1.5 GS-FLX plates for Ipomoea. littoralis, and 1 for Ipomoea. tenuissima)

The developmental stages for Ipomoea. littoralis and Ipomoea. tenuissima were:
1. Young, middle-aged and old leaves from pot-grown plants.
2. Young and old vines.
3. Young and old roots.
4. Mixed flower tissues.

Participants met at both locations several times during the year and have held conference calls for discussions on the project.